The ISME Journal (2017) 11, 1130–1141 © 2017 International Society for Microbial Ecology All rights reserved 1751-7362/17 www.nature.com/ismej ORIGINAL ARTICLE An acid-tolerant ammonia-oxidizing γ-proteobacterium from soil Masahito Hayatsu1, Kanako Tago1, Ikuo Uchiyama2, Atsushi Toyoda3, Yong Wang1, Yumi Shimomura1, Takashi Okubo1, Futoshi Kurisu4, Yuhei Hirono5, Kunihiko Nonaka5, Hiroko Akiyama1, Takehiko Itoh6 and Hideto Takami7 1Institute of Agro-Environmental Science, National Agriculture and Food Research Organization (NARO), Tsukuba, Ibaraki, Japan; 2National Institute for Basic Biology, Okazaki, Aichi, Japan; 3Center for Information Biology, National Institute of Genetics, Mishima, Shizuoka, Japan; 4Research Center for Water Environment Technology, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan; 5Institute of Fruit Tree and Tea Science, NARO, Shimada, Shizuoka, Japan; 6Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Meguro-ku, Tokyo, Japan and 7Yokohama Institute, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Yokohama, Kanagawa, Japan Nitrification, the microbial oxidation of ammonia to nitrate via nitrite, occurs in a wide range of acidic soils. However, the ammonia-oxidizing bacteria (AOB) that have been isolated from soil to date are acid-sensitive. Here we report the isolation and characterization of an acid-adapted AOB from an acidic agricultural soil. The isolated AOB, strain TAO100, is classified within the Gammaproteobac- teria based on phylogenetic characteristics. TAO100 can grow in the pH range of 5–7.5 and survive in highly acidic conditions until pH 2 by forming cell aggregates. Whereas all known gammaproteo- bacterial AOB (γ-AOB) species, which have been isolated from marine and saline aquatic environments, are halophiles, TAO100 is not phenotypically halophilic. Thus, TAO100 represents the first soil-originated and non-halophilic γ-AOB. The TAO100 genome is considerably smaller than those of other γ-AOB and lacks several genes associated with salt tolerance which are unnecessary for survival in soil. The ammonia monooxygenase subunit A gene of TAO100 and its transcript are higher in abundance than those of ammonia-oxidizing archaea and betaproteobacterial AOB in the strongly acidic soil. These results indicate that TAO100 plays an important role in the nitrification of acidic soils. Based on these results, we propose TAO100 as a novel species of a new genus, Candidatus Nitrosoglobus terrae. The ISME Journal (2017) 11, 1130–1141; doi:10.1038/ismej.2016.191; published online 10 January 2017 Introduction acid-sensitive and do not grow below pH 6.0 in pure culture (Jiang and Bakken, 1999). However, high- Extensive applications of nitrogen fertilizers have nitrification rates have been found in a wide variety enhanced nitrification activity, resulting in the of acidic soils with pH below 5.5 (Booth et al., 2005). pollution of surface and ground waters and emission Thus, the microbiological basis of ammonia oxida- of the greenhouse gas nitrous oxide (Schlesinger, tion in acidic soil remained unclear until the recent 2009; Wrage et al., 2001). Ammonia oxidation, the discovery of AOA (Konneke et al., 2005; Leininger first and rate-limiting step of nitrification, is carried et al., 2006; Tourna et al., 2011). Indeed, AOA are out in soil by ammonia oxidizing bacteria (AOB) more abundant than AOB in many acidic soils (Kowalchuk and Stephen, 2001), ammonia-oxidizing (Zhang et al., 2012), and acidophilic AOA, Nitroso- archaea (AOA) (Leininger et al., 2006) and complete talea devanaterra and Nitrosotalea species, have ammonia oxidizing (comammox) bacteria (Daims been isolated from acidic soil and characterized et al., 2015; van Kessel et al., 2015). AOB, of which (Lehtovirta-Morley et al., 2011, 2014). These obser- sequences have been found suggesting that they may vations strongly support the hypothesis that AOA be involved in ammonia oxidation in soil (Di et al., are more important than AOB in nitrification in 2009; Jia and Conrad, 2009), have been shown to be acidic soil. AOB have, however, been identified as the dominant ammonia oxidizers in several acidic soils Correspondence: M Hayatsu, Institute of Agro-Environmental (Long et al., 2012; Petersen et al., 2012; Wertz et al., Science, NARO, Tsukuba, Ibaraki 3058604, Japan. 2012). A few AOB have been isolated from acidic E-mail: [email protected] Received 12 May 2016; revised 16 November 2016; accepted soils with a pH around 4 (Jiang and Bakken, 1999) 19 November 2016; published online 10 January 2017 although they have been found to be acid sensitive in Ammonia-oxidizing γ-proteobacterium from soil M Hayatsu et al 1131 pure culture. Ammonia oxidation by these acid- Slurries were obtained by mixing 2.5 g soil sample sensitive AOB in acidic soils might be explained by with 10 ml of 1 mM potassium phosphate buffer (pH 7) several hypothetical mechanisms (De Boer and containing 10 mM (NH4)2SO4 and 10 mM KClO3. Kowalchuk, 2001) including pH-neutral micro-sites, Subsequently, the slurries were incubated for several urea hydrolysis (Burton and Prosser, 2001; De Boer hours at 25 °C with shaking at 150 r.p.m. Aliquots of et al., 1989), and acid resistant biofilm and aggregate 1 ml were drawn at 0 and 4 h and centrifuged at formation (Allison and Prosser, 1993; De Boer et al., 10000g for 10 min at 4 °C. The nitrite concentration 1991). Conversely, we previously reported the in the supernatant was determined calorimetrically potential presence of acid-tolerant AOB in acidic via diazotization (Keeney and Nelson, 1982). PAR soil from tea fields, where nitrification occurs at high were calculated as nmol nitrite per hour per g of levels despite the soil having a pH of about 3 dried soil. The obtained potential rates, which were (Hayatsu, 1993; Hayatsu and Kosuge, 1993). These measured at pH 7, may not reflect actual nitrification studies suggested the existence of acid adapted AOB rates in acidic soil at in situ pH. However, potential and their contribution to nitrification in acidic soil. nitrification rate assessment at this pH value has Therefore, AOB cannot be excluded from the organ- been utilized in many studies, including those on isms that mediate nitrification in acidic soil, and the nitrification in acidic soil (for example, Peterson discovery of new acid-tolerant AOB is anticipated. et al., 2012; Wertz et al., 2012). We used the potential The aim of the current study was to isolate and rate to facilitate comparisons with other studies. characterize acid-adapted AOB from the acidic soil of tea fields and to demonstrate the contribution of the isolated AOB to ammonia oxidation activity in Enrichment of ammonia oxidizing bacteria the tea field soil. In addition, we aimed to provide Soil samples with pH 2.9 and 3.1 were collected genomic level evidence for the taxonomic and from no Ca and high N treatment plots, respectively evolutionary relationships between isolated acid- (Table 1) and used to obtain enrichment cultures. adapted AOB and known related AOB. The initial cultures were obtained by suspending 10 g soil in 100 ml Schmidt AOB media (Schmidt and Belser, 1994) containing 100 mM ammonium Materials and methods sulfate at pH 5.5. These cultures were grown at 25 °C Soil samples with shaking at 110 r.p.m. until high-rates of ammo- Soil samples, classified as Andosol, were obtained in nia oxidation were observed. The cultures were then July 2012 from tea field plots at Kanaya Tea Research diluted 10-fold and grown further. Multiple cycles of Station, Institute of Fruit Tree and Tea Science, NARO, dilution-to-extinction were performed until no cul- in Japan. These plots are fertilized every year to study turable heterotrophic microorganisms were detected. the effects of N, P, K and Ca on tea plants (Table 1). N, Contaminating microorganisms were assessed by 10- P, K and Ca were supplied as ammonium sulfate, fold diluted nutrient agar. We designed 16S rRNA- superphosphate, potassium sulfate and dolomite specific fluorescent probes for fluorescence in situ hybridization (FISH) analysis and a polymerase (CaCO3·MgCO3), respectively. Soil samples were col- lected as cores from 10-cm deep and immediately chain reaction (PCR) primer set (Supplementary stored at − 80 °C until molecular analyses or at 4 °C Table S1) for enriched ammonia oxidizing bacteria until potential nitrification rate analyses. using retrieved 16S rRNA gene sequences of DNA fragments amplified from the enriched culture using the Bacteria-specific primers 27F and 1492r Potential ammonia oxidation rate (PAR) (Lane, 1991). The final culture purity was evalu- Potential rates of ammonia oxidation were measured as ated by FISH (Supplementary Methods) and quanti- rates of nitrite accumulation in the presence of chlorate, tative PCR (qPCR) for the 16S rRNA gene using which inhibits nitrite oxidation (Belser and Mays, 1980). the designed FISH probe and PCR primer set, Table 1 Characteristics of soils used in the study Treatment a Soil pH Total C (%) Total N (%) PARb Fertilization (kgha −1y− 1)c (nmole −1h−1g − 1) N input Ca (dolomite) Standard 3.36 (0.05) c 16.86 (1.32) b 1.23 (0.13) b 45.91 (5.74) a 400 1 000 High Ca 4.74 (0.10) d 11.93 (0.13) a 0.84 (0.04) a 253.66 (5.10) c 400 3 000 No Ca 2.89 (0.05) a 19.47 (0.59) c 1.54 (0.06) d 55.84 (4.80) a 400 0 High N 3.09 (0.04) b 17.82 (0.37) b 1.52 (0.06) c 180.18 (17.90) b 1200 1 000 s.d. are given in parentheses. Values within the same column followed by the same letter are not significantly different at Po0.05. aTreatments: standard fertilization (Standard), high-rate Ca fertilizer input (high Ca), no Ca fertilizer input (no Ca) and high-rate N fertilizer input (high N).